大鼠肝tRNA~(Ile)基因的克隆与体外转录

采用PCR扩增出大鼠肝tRNAIle基因,构建了重组质粒pGWIW,并用T7RNA聚合酶／启动子系统对其进行了体外表达．经过对转录产物片段大小及运用Northernblot鉴定,证明获得了不含修饰核苷酸的大鼠肝tRNAIle生物学活性检测显示：合成基因体外转录产物氨基酰化活性是天然tRNA的40％,提示修饰核苷酸在哺乳动物Ile－tRNA合成酶的识别过程中起重要作用     

用T7 RNA聚合酶体外转录合成大鼠肝tRNAIle

采用PCR技术从rec-M13mp18中扩增出120 bp的大鼠肝tRNA^Ile合成基因片段,经限制性内切酶BstNⅠ酶切后作为模板,利用T7 RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNA^Ile基因,生成不含修饰碱基的tRNA^Ile,并对体外转录反应条件进行了优化,回收的tRNA产量可达DNA模板量的40倍.     

肝癌细胞AFP基因高水平的转录是受核蛋白成分的促进

利用大鼠甲胎蛋白(AFP)基因片段作为模板,分析大鼠肝癌细胞核蛋白成分对体外转录活性的影响,发现大鼠肝癌含有促进AFP基因体外转录的核蛋白。作为对照．没有发现任何成年大鼠肝核蛋白可以促进AFP基因的体外转录。为了确定促进AFP基因体外转录的核蛋白作用部位,对AFP基因模板5＇端上游序列进行了不同程度的删除,进一步分析核蛋白对删掉5’端上游序列后的模板体外转录的影响,结果表明,AFP基因转录的起始点到255bp这段DNA序列是大鼠肝癌核蛋白促进AFP基因转录必不可少的。以SV40DNA经Pst I酶酶切所得的DNA片段(1216bp和4027bp)代替AFP基因片段作为模板,不存在核蛋白促进体外转录的现象。用AFP基因转录的起始点到 255bp这段的DNA为探针,进行Southwestm印迹分析,结果发现了8种与探针结合的核蛋白。     

斑马鱼Gfi1.1基因的克隆及其体外转录

目的:克隆斑马鱼Gfi1.1基因的全长cDNA,运用T7 RNA聚合酶对含有Gfi1.1基因的ORF区进行体外转录,在体外合成5端带有帽子结构的Gfi1.1 mRNA分子,为后续研究斑马鱼Gfi1.1基因的功能打下基础。方法:应用RT-PCR从斑马鱼组织中扩增出Gfi1.1 cDNA片段,经回收纯化与pGM-T载体连接并转化感受态细菌DH-5α,通过蓝白筛选酶切鉴定阳性菌落,小量提取质粒,Nde I限制性内切酶线性化pGM-T-Gfi1.1质粒,运用T7 RNA聚合酶对Gfi1.1基因进行体外转录及加帽。经凝胶电泳对目的片段进行鉴定。结果:RT-PCR扩增获得约1.2 kb的DNA片段,DNA序列分析的结果与GenBank上的序列(NM_001020776)一致,酶切线性化及体外转录加帽pGM-T-Gfi1.1,凝胶电泳鉴定RNA分子大小与预期完全一致。结论:成功克隆斑马鱼Gfi1.1基因并体外转录及加帽pGM-T-Gfi1.1。     

rmf基因启动子识别因子选择性研究

分别用σ^(70)或σ^(38)因子和核心酶(Core enzyme)重建RNA聚合酶全酶(Holoenzyme)对含有rmf基因启动子的DNA模板进行体外转录。不同的限制性内切酶酶切片段模板证实了rmf基因转录起始位点,尽管rmf基因在大肠杆菌进入稳定期大量表达,但是其启动子对稳定期起重要作用的σ^(38)因子的识别能力很低,而只能被σ^(70)所识别。rmf基因启动子体外转录的最适温度为37℃,最适NaCl浓度为50mmol/L。     

大肠杆菌等受体tRNALeu的T7 RNA聚合酶转录

用化学方法合成编码2个大肠杆菌tRNALeu(tRNALeu1和tRNALeu2)的基因和T7启动子,分别克隆到pUC19载体上,并在纯化的T7 RNA聚合酶的体外转录系统中转录出不含修饰核苷酸的tRNALeu.在T7转录体系中,亚精胺对转录有负影响.在最适转录条件下,可以得到有活力的RNA转录物的量是模板DNA的250倍左右.在大肠杆菌亮氨酰-tRNA合成酶的催化下,2种经体外转录产生的未修饰等受体tRNALeu(tRNALeu1和tRNALeu2)的亮氨酸接受能力基本相同,但只有从体内纯化对应的tRNALeu的四分之一左右,表明修饰核苷酸在tRNALeu氨酰化过程中起着较为重要但非关键的作用.     

利用PCR技术构建体外高效转录系统

设计并合成了一对 PCR 反应引物,其5′端引物除含有目的基因5′端序列外,还外加 T7 RNA 聚合酶启动子的17个核苷酸.3′端引物则按常规设计.以染色体 DNA为模板,通过 PCR,可扩增出带有 T7 RNA 聚合酶启动子的目的基因 DNA 片段.以此 PCR 产物为模板,在体外成功实现了高效转录.这是一种快速、简便构建体外高效转录系统的好方法.     

siRNA抑制丙型肝炎病毒IRES介导的基因表达

以HCVIRES为靶位 ,应用T7RNA多聚酶体外转录合成了 5条小干扰RNA(siRNA) .脂质体转染法将其导入HCVIRES介导萤光素酶表达的转基因细胞 (HepG2 .970 6 )中 ,通过测定萤光素酶的量 ,评价T7siRNA对HCV介导基因表达的抑制作用 .结果表明 ,所合成的 5条T7siRNAs ,均能特异性地抑制萤光素酶基因的表达 ,抑制率分别为 94 31%、80 0 1%、78 0 1%、80 33%、85 6 4% ,其中以靶向HCVIRES第二茎环结构的T7siRNA1抑制率最高 ,且对HCV基因的抑制作用有剂量依赖性 ,随T7siRNA1量的增加 ,抑制率逐渐增强 .siRNA抑制HCV基因的作用具有良好的特异性 ,改变其中 1个核苷酸即无显著抑制作用 .     

Mip1对大鼠心肌细胞凋亡基因Bid转录调控作用的初探

目的:研究转录因子Mip1对大鼠心肌细胞H9C2凋亡相关基因Bid的转录调控作用.方法:以H9C2细胞基因组DNA为模板,扩增Bid核心启动子片段,将其克隆入荧光素酶报告基因质粒PGL3-Basic中,构建重组载体,并采用双酶切法、PCR法及基因测序对其进行鉴定.用脂质体转染法将该载体转入Mip1不同程度过表达的H9C2细胞,检测该细胞Bid基因启动子区的转录活性.结果:成功克隆Bid基因启动子区,双酶切、PCR和基因测序均显示PGL3-Basic-Bid promoter重组载体构建成功.荧光素酶相对活性检测显示在H9C2细胞中,随着Mip1转入的增多,Bid启动子区转录活性逐渐下降.结论:Mip1作为一个新的转录抑制因子,可以下调凋亡基因Bid的转录.     

银松素合酶基因地高辛标记RNA探针的合成

目的:体外转录合成银松素合酶(PS)基因地高辛标记反义RNA探针。方法:通过双酶切把PScDNA全长序列接到pBlueskriptⅡKS( )的多克隆位点上;同时,经由NCBI比对确定PScDNA的特异序列,设计含有SalⅠ和XbaⅠ这2个酶切位点的上、下游引物,用PCR法扩增含有该位点的目的片段,并连接到pBlueskriptⅡKS( )的多克隆位点上;最后利用pBlueskriptⅡKS( )上的启动子T7,用T7RNA聚合酶在体外转录合成地高辛标记PS全长及特异序列的反义RNA探针。结果:通过对构建探针的单、双酶切,PCR及电泳鉴定,表明成功合成了PS基因地高辛标记的全长及特异序列的反义RNA探针。结论:RNA探针的合成为后续马尾松PS基因表达的RNA原位杂交研究打下了基础。     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

为《Flora of China》提供的伞形科新资料

在为编写《Flora of China》伞形科而进行的修订工作中,提出了11个新组合,即矮小丝瓣芹Acronema minus (M. F. Watson) M. F. Watson & Z. H. Pan, 短柄丝瓣芹A. brevipedicellatum Z. H. Pan & M. F. Watson, 川西当归Angelica sinensis var. wilsonii (H. Wolff) Z. H. Pan & M. F. Watson, 云南细裂芹Harrysmithia franchetii (M. Hiroe) M. L. Sheh, 钝叶独活Heracleum candicans var. obtusifolium (Wall. ex DC.) F. T. Pu & M. F. Watson, 中华天胡荽Hydrocotyle hookeri ssp. chinensis (Dunn ex R. H. Shan & S. L. Liou) M. F. Watson & M. L. Sheh, 普渡天胡荽H. hookeri ssp. handelii (H. Wolff) M. F. Watson & M. L. Sheh, 锐棱岩风Libanotis grubovii (V. M. Vinogradova) M. L. Sheh & M. F. Watson, 美脉藁本Ligusticum likiangense (H. Wolff) F. T. Pu & M. F. Watson和线叶藁本L. nematophyllum (Pimenov & Kljuykov) F. T. Pu & M. F. Watson, 无管藁本L. nullivittatum (K. T. Fu) F. T. Pu & M. F. Watson和二色棱子芹Pleurospermum bicolor (Franch.) C. Norman ex Z. H. Pan & M. F. Watson.; 发现了1个新种,即短柄丝瓣芹。此外,还为Pleurospermum govanianum var. bicolor Franch.指定了后选模式。     

Entomopathogenic Nematode Infectivity Assay: Comparison of Penetration Rate into Different Hosts

Penetration rate (the percentage of the initial infective juvenile inoculum that invades an insect host) was tested as an indicator of entomopathogenic nematode infectivity. Several host-parasite-substrate combinations were evaluated for penetration rate. Four steinernematids, Steinernema carpocapsae, S. glaseri, S. feltiae, S. riobravis and two strains of Heterorhabditis bacteriophora were tested in a contact bioassay against the wax moth, Galleria mellonella, the yellow meal worm, Tenebrio molitor, the beet armyworm, Spodoptera exigua, the black cutworm, Agrotis ipsilon, and the European corn borer, Ostrinia nubilalis. The insect larvae were confined individually in sand and filter paper arenas and exposed to 200 infective juveniles. After incubation, dead insects were dissected in order to count the nematodes penetrated. The data were analyzed for the effects of nematode strain and substrate on penetration rate. The bioassay substrate had a variable effect depending on the insect species. The nematode effect was highly significant for all insects tested. The penetration rate therefore allowed comparisons among nematode strains invading a host. Nematode ranking for infectivity differed according to the insect tested.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

The Role of Parasitoids and Entomopathogenic Fungi in Mortality of Third-instar and Adult Ceroplastes destructor and C. sinensis (Hemiptera: Coccidae: Ceroplastinae) on Citrus in New Zealand

The incidence of parasitism and disease in third-instar and adult Ceroplastes destructor Newstead and C. sinensis Del Guercio (Hemiptera: Coccidae) on citrus in Northland, New Zealand, was measured from 1991-1994. Both species were parasitized by female Euxanthellus philippiae Silvestri (Hymenoptera: Aphelinidae). Female E. philippiae were hyper-parasitized by males of their own species and by Coccidoctonus dubius Girault (Hymenoptera: Encyrtidae). Male E. philippiae were also tertiary parasitoids of C. dubius. E. philippiae was found in third-instar and adult C. sinensis , but was virtually restricted to third-instar C. destructor . Parasitoid phenology varied according to the instar and species of the host. Two fungal pathogens, Verticillium lecanii (Zimmermann) Viegas and Fusarium spp., were identified from both C. destructor and C. sinensis . Disease was a greater mortality factor than parasitism in C. destructor , whereas the opposite applied to those C. sinensis for which the cause of death could be identified. Disease levels were underestimated, particularly in C. destructor. Mortality from parasitism and disease did not act in a density-dependent manner.     

Allozyme relationships among ten species of Rhodniini, showing paraphyly of Rhodnius including Psammolestes

Genetic relationships among 10 species of bugs belonging to the tribe Rhodniini (Hemiptera: Reduviidae), including some important vectors of Chagas disease, were inferred from allozyme analysis of 12 enzyme loci (out of 21 enzyme systems examined), using agarose gel electrophoresis. These species formed two clusters: one comprising Rhodnius brethesi, R. ecuadoriensis, R. pallescens and R. pictipes; the other with Psammolestes tertius, Rhodnius domesticus and the Rhodnius prolixus group comprising R. nasutus, R. neglectus, R. prolixus and R. robustus. The resulting tree was [((R. ecuadoriensis, R. pallescens) R. brethesi) R. pictipes], [R. domesticus (P. tertius [(R. nasutus, R. neglectus) (R. prolixus, R. robustus)])]. Rhodnius nasutus and R. neglectus differed by only one locus, whereas no diagnostic loci were detected between R. prolixus and R. robustus (22 loci were analysed for these four species), despite considerable DNA sequence divergence between species in each of these pairs. Allozymes of the R. prolixus group showed greater similarity with Psammolestes tertius than with other Rhodnius spp., indicating that Rhodnius is paraphyletic and might include Psammolestes.     

Description d'une nouvelle espèce et analyse morpho-fonctionnelle du genre Doryaspis White (Heterostraci) du Dévonien du Spitsberg

Doryaspis groenhorgensis nov. sp. is a new pteraspidiform from the lower devonian of Spitsbergen. The genus Doryaspis is now considered as the most abundant pteraspidiform of the Wood Bay formation, with five described species. Moreover, the pteraspidiform diversity of this formation has been under rated all along the XXth century. A morpho-functional analysis of the Doryaspis generic characters (e.g. flat dorsal shield, ventral pseudorostrum, long cornual plates) allows to consider two possible mode of life. However, none of the pelagic or benthic mode of life is better supported than the other. The same analysis introduce some interpretative hypothesis on histology and moving of the Pteraspidiformes. The Pteraspidiformes diversity of Spitsbergen is important for further Devonian circum-arctic comparisons (e.g. siberian platform).     

Seasonal and Habitat Variability in the Fungal Pathogens, Neozygites cf. floridana and Hirsutella thompsonii, Associated with Cassava Mites in Benin, West Africa

A survey of the pathogenic fungi associated with mites on cassava in Benin, West Africa, revealed both geographical and seasonal variation in the presence of Neozygites cf. floridana (Weiser and Muma) and Hirsutella thompsonii Fisher on Mononychellus tanajoa (Bondar) and Oligonychus gossypii (Zacher). Few dead and infected mites were found during the dry season, regardless of vegetation zone. In three of 30 surveyed sites, N. floridana was found infecting 1% of the dead M. tanajoa and 2% of the dead O. gossypii, while H. thompsonii was observed infecting 20% of the dead M. tanajoa in a single site. The frequency of sites having infected mites during the wet season was 3.5 times greater than that seen during the dry season. N. floridana infected 10% of the dead M. tanajoa and 19% of the dead O. gossypii on young leaves. Mites infected with N. floridana were found either in the coastal Southern Forest Mosaic (SFM) or in the Northern Guinea Savanna vegetation zones. N. floridana was rare in the low mite densities associated with mature leaves. H. thompsonii was found on 19% and 29% of the dead M. tanajoa on young and mature leaves respectively. All M. tanajoa infected with H. thompsonii on young leaves and mature leaves (75%) were found in the SFM. A single M. tanajoa was the only infected mite found in the Southern Guinea Savanna. Relatively few O. gossypii were infected with H. thompsonii. N. floridana and H. thompsonii were found together in three sites, but never on the same host. Phytoseiids were never found infected with either pathogen. In a regression analysis, the number of dead mites was significantly estimated from the total number of mites for both species, regardless of leaf age. The numbers of dead M. tanajoa on mature leaves were also estimated from the proportion infected with H. thompsonii. The numbers of infected mites on young leaves were estimated from their association with the SFM for M. tanajoa infected with H. thompsonii, and from total mites for O. gossypii infected with N. floridana. On mature leaves, infected mite numbers were estimated from the numbers of dead M. tanajoa infected with H. thompsonii. The merit of introducing more virulent or better adapted isolates of N. floridana to control M. tanajoa in Africa is discussed.     

Materials for Chinese Phyllanthoideae

The present paper is part of taxonomic study on Chinese Phyllanthoideae. Included in it are two new varieties, Leptopus esquirolii var. villosus and Drypetes hainanensis var. longistipitata, one new combination, Glochidion triandrum var. siamense, and seven new records in China: Drypetes salicifolia, D. hoaensis. Actephila subsessilis, Glochidion khasicum, G. nubigennum, Bridelia spiosa and B. poilanei. In addition, seventeen taxon names are newly reduced: Liodendron formosanum = Drypetes formosana, Liodendron matsumurae = Drypetes matsumurae, D. longipes = D. indica, Antidesma paxii = A. acidum, A. hiiranense, A. filipes and A. pentandrum var. hiiranense = A. japonicum, A. calvescens = A. montanum, A. microphyllum = A. venosum, Breynia stipitata var. formosana and B. jormosana = B. vitis-idaea, Glochidion zeylanicum var. tomentosum = G. hirsutum, G. rubidulum = G. thomsonii, G. acuminatum = G. triandrum, G. fagifolium and Phyllanthus fagifolius = Glochidion sphaerogynum, Bridelia penangiena = B. insulana, B. henryana = B. tomentosa. All the types are kept in SCBl and PE.